The human genome contains many at-risk sequences that are prone to mutations including diverse repeated sequences, segmental duplications and regions of copy number variations. Such repetitive sequence elements can cause genome rearrangements through non-allelic homologous recombination (HR). Many human diseases are known to be caused by chromosomal rearrangements mediated by non-allelic HR. Moreover, many cancers exhibit ongoing genome rearrangements, stimulated by numerous at-risk sequences in the genome. The yeast Saccharomyces cerevisiae provides a powerful model system to study genome rearrangements. Despite that many pathways have been found to suppress genome rearrangements, the understanding of how duplication-mediated genome rearrangements are specifically suppressed remains rudimentary. Sumoylation is an evolutionarily conserved post-translational modification of proteins and it is known to regulate many nuclear activities includin gene transcription, chromosome segregation, DNA replication and repair. We have discovered a new function of protein sumoylation in suppression of duplication- mediated genome rearrangement and found that the SUMO pathway is by far the most important pathway in preventing this type of genome rearrangements. In the proposed studies, we will characterize the genetics and biochemistry of the SUMO pathway focusing on its function in genome maintenance. We will pursue the following specific aims: First, we will characterize the role of essential genes in genome maintenance. Second, we will determine the enzyme-substrate relationship in the SUMO pathway, focusing on the function and substrates of SUMO-specific isopeptidase. Third, we will study how sumoylation of Mms21-specific substrates contribute to genome maintenance. In all of our studies, we will employ a combination of genetic, biochemical and proteomic approaches to study how the SUMO pathway specifically prevents duplication-mediated genome rearrangements, which are expected to provide new insights into the genetic basis of human cancers.